The long-term goal of the proposed research is to elucidate the mechanism of host innate immune responses and viral counter-defense responses. RNAi refers to the gene regulatory mechanism guided by small RNAs and is highly conserved from plants, invertebrates to humans. In 2002 we provided the first direct evidence for a natural antiviral role of RNAi in animals using a Drosophila cell culture model. Subsequently we showed that positive-strand RNA virus replication also triggers the RNAi immunity in mosquitoes and nematodes, and established the first molecular framework for the new viral immunity mediated by the dsRNA-siRNA pathway of RNAi in adult Drosophila. Recent studies from others indicate that RNAi also plays an important role in mammalian responses to viruses. As a counter-defense, viruses including those infecting humans encode proteins capable of RNAi suppression, referred to as viral suppressors of RNAi (VSR). The VSR activity of flock house virus B2 protein in preventing viral dsRNA from dicing into siRNAs is essential for infection because B2 expression becomes dispensable in infection of RNAi-defective Drosophila mutants. However, such a specific role in RNAi suppression during virus infection has not been established for any other animal VSR. Aim 1 will determine if active RNAi suppression is required for infection by the polio-like cricket paralysis virus, and test the hypothesis that serial passage of viruses in RNAi-defective mutant flies would result in the accumulation of loss-of-function mutations within their VSR genes because of elimination of the viral immunity. Small RNAs determine the specificity of RNAi mechanism and their origin reveals the identity of RNAi inducer. Drosophila produces small-interfering RNAs (siRNA), microRNAs (miRNA) and repeat- associated siRNAs (rasiRNA) in distinct genetic pathways. Aim 2 will clone, sequence, and investigate the biogenesis and antiviral activities of virus-derived siRNAs in infected Drosophila, determine if miRNA and/or rasiRNA pathways also participate in the viral immunity, and characterize new components of the immunity identified in a genome-wide screen. Furthermore, diverse VSRs that target distinct steps in the RNAi pathway have been identified. In contrast, little is known about if and how host organisms regulate the viral VSR activities. Aim 3 will test the hypothesis that serine phosphorylation and lysine ubiquitination we detected in the B2 protein expressed in infected Drosophila cells represent a new host defense strategy to destroy VSR in a phosphorylation-dependent proteasome pathway. Fruit fly has been a powerful model for elucidating the molecular mechanisms of both innate immunity and RNAi in humans. It is likely that the proposed studies on Drosophila immune responses to infection by model positive-strand RNA viruses and the viral counter- defensive strategies will provide mechanistic insights into the molecular interactions between viruses and mammalian hosts.